Immunogenic compositions comprising dimeric forms of the human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) envelope glycoproteins

ABSTRACT

The invention relates to an isolated immune complex comprising a protein and an antibody that binds with said protein, wherein the protein is selected from the group consisting of gp80 of HIV-2 and gp65 of SIV, wherein said gp80 is a glycoprotein having an apparent molecular weight of 80 kDa, as determined by SDS-PAGE, and further wherein said gp65 is a glycoprotein having an apparent molecular weight of 65 kDa as determined by SDS-PAGE. Also provided are an immunogenic composition comprising an amount of gp80 protein of human immunodeficiency virus type 2 (HIV-2) sufficient to induce an immune response and a pharmaceutically acceptible carrier, and a composition comprising at least one antigen selected from the group consisting of gp80 protein of HIV-2 and gp65 SIV .

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/002,756, filed Jan. 13,1993, now U.S. Pat. No. 5,470,702 which is a division of applicationSer. No. 07/356,459, filed May 25, 1989, now U.S. Pat. No. 5,208,321,which is a continuation-in-part of application Ser. No. 07/204,346,filed Jun. 9, 1988, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to viral proteins and glycoproteins, tocompositions containing these proteins, to methods of preparing theproteins, and to their use in detecting viral inection.

Human immunodeficiency virus (HIV) is the etiological agent of acquiredimmunodeficiency syndrome (AIDS) (Montagnier et al., 1984). To date, tworelated but distinct viruses HIV-1 and HIV-2, have been identified(Barre-Sinoussi et al., 1983; Brun-Vezinet et al., 1987; Clavel et al.,1986a, 1986b; Guyader et al., 1987; Popovic et al., 1984; Ratner et al.,1985; Wain-Hobson et al., 1985). HIV-2 is closely related to simianimmunodeficiency virus (SIV-mac), which causes an AIDS-like disease inmacaques (Daniel et al., 1985; Fultz et al., 1986; Chakrabarti et al.,1987). Alignments of the nucleotide sequences of HIV-1, HIV-2, and SIVreveal a considerable homology between HIV-2 and SIV-mac. These twoviruses share about 75% overall nucleotide sequence homology, but bothof them are only distantly related to HIV-1 with about 40% overallhomology (Guyader et al., 1987; Chakrabarti et al., 1977).

In addition to the genes that encode structural proteins (the virioncapsid and envelope glycoproteins) and the enzymes required for proviralsynthesis and integration common to all retroviruses, HIV-1, HIV-2, andSIV encode genes that regulate virus replication as well as genes thatencode proteins of yet unknown function. The only notable difference inthe genetic organizations of HIV-1, HIV-2, and SIV resides in the openreading frame referred to as vpx, which is absent in HIV-1 and vpu inHIV-1 but not in HIV-2 and SIV (Cohen et al., 1988; Guyader et al.,1987). These viruses are both tropic and cytopathic for CD4 positive Tlymphocytes (Klatzmann et al., 1984; Clavel et al., 1985a; Dalgleish etal., 1984; Daniel et al., 1985). A great number of studies haveindicated that CD4 functions as the cellular receptor of HIV (Weiss,1988).

The HIV-1 env gene encodes a 160-kilodalton (kDa) glycoprotein that isproteolytically cleaved to yield the extracellular and transmembraneproteins, gp120 and gp41, respectively (Montagnier et al., 1985).Similarly, HIV-2 env gene encodes a precursor glycoprotein which is thenprocessed to the mature extracellular and transmembrane glycoproteins(Rey et al., 1989). However, unlike HIV-1, the processing of HIV-2envelope precursor gp140 seems to require the formation of a homologousdimer (gp300) during its processing. Interestingly, dimerization of theenvelope precursor is also observed in SIV infected cells (Rey et al.1989). Accordingly, dimer formation seems to be a specific property ofHIV-2 and SIV envelope gene expression.

There exists a need in the art for additional information on thestructure and in vivo processing of HIV-2 proteins, and especially HIV-2envelope proteins and glycoproteins. Such information would aid inidentifying HIV-2 infection in individuals. In addition, such findingscould aid in elucidating the mechanism by which HIV-2 infection andvirus proliferation occur and thereby make it possible to devise modesof intervening in viral processes.

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art by providingHIV-2 envelope proteins and glycoproteins in purified form. Moreparticularly, this invention relates to the processing of HIV-2 envelopeglycoproteins and the characterization of the transmembraneglycoprotein. Previously, the detection of the transmembraneglycoprotein had been handicapped by the lack of specific antibodies.For this reason, polyclonal antibodies were prepared against thepurified HIV-2 envelope precursor. Furthermore, monoclonal antibodieswere prepared against a synthetic polypeptide deduced from the sequenceof the transmembrane glycoprotein of HIV-2. With the help of theseantibodies the membrane glycoproteins of HIV-2 and SIV were identified.

It was discovered that the transmembrane proteins exist as a homodimerin the infected cells as well as in the virions. Dimeric forms of thetransmembrane giycoproteins of HIV-2 and SIV can be dissociated in anionic detergent to 36 kDa and 32 kDa proteins, respectively.Conformational modifications brought about by the formation of envelopeprecursor might be necessary for transport of the glycoprotein precursorto the Golgi apparatus and its processing into the mature glycoproteinproducts, the extracellular and transmembrane envelope proteins.Furthermore, the transmembrane dimer might be essential for optimalstructure of the virion and thus its infectivity.

This invention thus provides gp80 structural glycoprotein of HIV-2dimeric form of the transmembrane glycoprotein and human retroviralvariants of HIV-2 containing the structural glycoprotein in purifiedform.

A similar high molecular weight glycoprotein of Simian ImmunodeficiencyVirus (SIV) or of a Simian retroviral variant of SIV has also beendiscovered. This glycoprotein is the dimeric form of transmembraneglycoprotein of SIV and has an apparent molecular weight of about 65 kDa(gp65_(SIV)). This glycoprotein is also provided in a purified form.

This invention also provides labeled gp80 of HIV-2 and gp65 of SIV.Preferably, the labeled glycoproteins are in purified form. It is alsopreferred that the labeled glycoprotein is capable of beingimmunologically recognized by human body fluid containing antibodies toHIV-2 or SIV. The glycoproteins can be labeled, for example, with animmunoassay label selected from the group consisting of radioactive,enzymatic, fluorescent, chemiluminescent labels, and chromophores.

Immunological complexes between the proteins and glycoproteins of theinvention and antibodies recognizing the proteins and giycoproteins arealso provided. The immunological complexes can be labeled with animmunoassay label selected from the group consisting of radioactive,enzymatic, fluorescent, chemiluminescent labels, and chromophores.

Furthermore, this invention provides a method for detecting infection ofcells by human immunodeficiency virus type-2 (HIV-2). The methodcomprises providing a composition comprising cells suspected of beinginfected with HIV-2, disrupting cells in the composition to exposeintracellular proteins, and assaying the exposed intracellular proteinsfor the presence of gp80 glycoprotein of HIV-2. The exposedintracellular proteins are typically assayed by electrophoresis or byimmunoassay with antibodies that are immunologically reactive with gp80glycoprotein of HIV-2.

This invention provides still another method of detecting antigens ofHIV-2, which comprises providing a composition suspected of containingantigens of HIV-2, and assaying the composition for the presence of gp80glycoprotein of HIV-2. The composition is typically free of cellulardebris. The molecular weight of the gp80 is estimated more or less 10%.The same for the other molecular weight mentioned in the presentinvention.

A method of distinguishing HIV-2 infection from HIV-1 infection in cellssuspected of being infected therewith has also been discovered. Themethod comprises providing an extract containig intracellular proteinsof the cells, and assaying the extract or the presence of gp80glycoprotein. The gp80 is characteristic or HIV-2, but the glycoproteinhas not been found in extracts of HIV-1 cell cultures.

In addition, this invention provides a method of making gp80glycoprotein of HIV-2, which comprises providing a compositioncontaining cells in which HIV-2 is capable of replicating, infecting thecells with HIV-2, and culturing the cells under conditions to causeHIV-2 to proliferate. The cells are then disrupted to exposeintracellular proteins. The gp80 glycoprotein is recovered from theresulting exposed intracellular proteins. It could be also recovered bydetergent solubilization of HIV-2 virions.

This invention also provides an in vitro diagnostic method for thedetection of the presence or absence of antibodies which bind to anantigen comprising the proteins or glycoproteins of the invention ormixtures of the proteins and glycoproteins. The method comprisescontacting the antigen with a biological fluid for a time and underconditions sufficent for the antigen and antibodies in the biologicalfluid to form an antigen-antibody complex, and then detecting theformation of the complex. The detecting step can further comprisemeasuring the formation of the antigen-antibody complex. The formationof the antigen-antibody-complex is preferably measured by immunoassaybased on Western Blot technique, ELISA (enzyme linked immunosorbentassay), indirect immunofluorescent assay, or immunoprecipitation assay.

A diagnostic kit for the detection of the presence or absence ofantibodies which bind to the proteins or glycoproteins of the inventionor mixtures of the proteins and glycoproteins contains antigencomprising the proteins, glycoproteins, or mixtures thereof and meansfor detecting the formation of immune complex between the antigen andantibodies. The antigens and the means are present in an amountsufficient to perform the detection.

This invention provides a method of preparing envelope transmembraneglycoproteins, which comprises providing an extracellular compositioncontaining gp80 glycoprotein of HIV-2 or gp65 of SIV and thendissociating the glycoprotein of HIV-2 or the glycoprotein of SIV. Anon-glycosylated dimeric form of the transmembrane envelope protein ofHIV-2 (and SIV) can be obtained from the glycosylated form of gp80 (orgp65) by using specific enzymes (i.e. endo F), which cleave matureoligosaccharide chains. Another procedure for the production ofunglycosylated forms of such dimeric protein could be geneticengineering methods (see Reference 16).

This invention also provides an immunogenic composition comprising aprotein or glycoprotein of the invention in an amount sufficient toinduce an immunogenic or protective response in vivo, in associationwith a pharmaceutically acceptable carrier therefor. A vaccinecomposition of the invention comprises a utralizing amount of thedimeric transmembrane envelope glycoprotein or unglycosylated formthereof and a pharmaceutically acceptable carrier therefor.

The dimeric form of the transmembrane glycoprotein is highly recognizedby all sera positive for HIV-2 antigens. Therefore, the detection ofgp80 could be used as a marker for characterization of HIV-2 positivesera and for differentiation from HIV-1 positive sera.

The proteins and glycoprotein of this invention are thus useful as aportion of a diagnostic composition for detecting the presence ofantibodies to antigenic proteins associated with HIV-2 and SIV. Inaddition, the proteins and glycoproteins can be used to raise antibodiesfor detecting the presence of antigenic proteins associated with HIV-2and SIV. The proteins and glycoproteins of the invention can be alsoemployed to raise neutralizing antibodies that either inactivate thevirus, reduce the viability of the virus in vivo, or inhibit or preventviral replication. The ability to elicit virus-neutralizing antibodiesis especially important when the proteins and glycoproteins of theinvention are used in vaccinating compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described in greater detail by referring to thedrawings in which:

FIG. 1 is an autoradiograph of a specific 80-kDa protein in HIV-2infected cells. Western blot analysis was made using an HIV-1 positiveserum and 3 HIV-2 positive sera (A, B and C). Extracts (material from10⁶ cells) from control uninfected (lanes 2), HIV-1 infected (lanes 1),and HIV-2 infected (lanes 3) CEM cells were analyzed by polyacrylamidegel (7.5%) electrophoresis before the Western blot assay. On the left,the arrows indicate the position of HIV-1 extracellular glycoprotein(gp120) and gaq precursors p55 and p40. On the right is the position ofHIV-2 specific gp300, gp140, and gp80 .

FIG. 2 is a fluorograph relating to synthesis of HIV-2 relatedglycoproteins. HIV-2 infected cells were labeled with [³H] glucosamine(200 μCi/ml; 4×10⁶ cells/ml) for 2, 3, 4, 6, and 8 hr. At differentpoints, extracts (material from 10⁷ cells) were prepared from infectedcells and from the virus pellet (prepared by 100,000 g centrifugation ofthe culture medium). Aliquots from these extracts (corresponding to2×10⁶ cells) were purified on HIV-2 serum-Sepharose, and the labeledproteins were analyzed by polyacrylamide gel (12.5%) electrophoresis. Onthe left is the position of protein mol. wt. markers: myosin, 200,000;phosphorylase B, 97,000; bovine serum albumin, 68,000; ovalbumin,43,000; carbonic anhydrase, 30,000.

FIG. 3 depicts a Western blot analysis with polyclonal antibodiesagainst gp300. On the left, extracts from uninfected (−) and HIV-1 orHIV-2 infected CEM cells are shown. On the right, extracts from HIV-2infected CEM cells are shown: cell extracts (lane C) and virus pellet(lane V). These samples were analyzed by polyacrylamide gelelectrophoresis (7.5% gel on the left; 12.5% gel on the right) beforeWestern blot analysis using murine polyclonal antibodies raised againstthe purified gp300 (anti-gp300). An autoradiogram is shown. Each samplecorresponded to material from 10⁶ cells.

FIG. 4 depicts a Western blot analysis using the monoclonal antibody mAb1H8. Extracts from HIV-1 or HIV-2 infected cells (lanes C) and viruspellet (lanes V) were analyzed by polyacrylamide gel (12.5%)electrophoresis before Western blot assay using mAb 1H8. Anautoradiograph is shown. This monoclonal antibody was raised againstsynthetic peptide p39′ of the purified HIV-2 virus and it is directedagainst the transmembrane glycoprotein of HIV-2 envelope.

FIG. 5 shows that peptide p39′ blocks the binding of mAb 1H8 to gp80.Extracts from the HIV-2 virus pellets were analyzed by Western blotassay using mAb 1H8 (section mAb) or anti-gp300 polyclonal antibodies(section S). Incubation with each antibody was carried out in theabsence (lanes−) or presence (lanes+) of 10 μg of peptide p39′. Theresults of the autoradiography are shown.

FIG. 6 depicts the results of pulse chase experiments to show theproduction of gp80 in HIV-2 infected cells. HIV-2 infected CEM cellswere labeled with [³⁵S] methionine (200 μCi/ml; 4×10⁶ cells/ml) for 2 hr(lanes 0). The radioactive label was then chased in culture mediumcontaining 5 mM cold methionine for 2 and 4 hr (lanes 2 and 4). Theculture medium at 4 hr was centrifuged at 100,000 g and the pellet wasextracted. All samples were immunoprecipitated using anti-gp300 or mAb1H8 antibodies. The labeled proteins in the immune complex preparationswere eluted in the electrophoresis sample buffer and analyzed byplyacrylamide gel (7.5%) electrophoresis. A fluorograph is shown. C andV stand for cell and virus extracts, respectively. Each samplerepresents material from 10⁶ cells.

FIG. 7 shows (a) incorporation of labeled glucosamine and fucose intogp80; and (b) the effect of castanospermine on the production of gp125and gp80. (a) HIV-2 infected CEM cells labeled with [³H] glucosamine(200 μCi/ml or with [³H] fucose (200 μCi/ml) were assayed byimmunoprecipitation using anti-gp300 polyclonal antibodies (lanes S) orthe monoclonal antibody mAb 1H8 (lanes M). All samples were analyzed bypolyacrylamide gel (12.5%) electrophoresis. A fluorograph is shown. (b)HIV-2 infected cells were labeled (16 hr) with [³⁵S] methionine (200μCi/ml; 4×10⁶ cells/ml) in the absence (lane−) or presence (lanes+) ofcastanospermine (1 mM). Extracts from the culture medium containingvirus particles were purified by immunoaffinity column using HIV-2serum-Sepharose, and the purified proteins were assayed bypolyacrylamide gel (12.5%) electrophoresis. A fluorograph is shown.

FIG. 8 relates to dissociation of gp80. Section C; extracts from [³⁵S]methionine labeled, HIV-2 infected CEM cells were assayed byimmunoprecipitation using the monoclonal antibody mAb 1H8 (lane 1).Another aliquot of the same cell extract preparation was first heated(95° C., 5 min) in the presence of 1% SDS, then it was diluted 10 foldin RIPA buffer before the immunoprecipitation assay (lane 2). The immunecomplex preparations were analyzed by electrophoresis. Each sample wasfrom extracts corresponding 10⁶ cells. Section V: HIV-2 virus pelletsfrom [³⁵S] methionine labeled cells (each corresponding to material from10⁷ cells) were suspended in different buffers: (1) lysis buffercontaining Triton (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1%(v/v) Triton X-100 and 100 units/ml aprotinin); (2) lysis buffercontaining SDS (as in 1, but containing 1% (v/v) SDS instead TritonX-100); (3) lysis buffer containing SDS and then heated (95° C., 5 min);(4) RIPA buffer (as in 1, but also containing 0.1% (v/v) SDS and 0.2%(v/v) deoxycholate). All these samples were then immunoprecipitatedusing mAb 1H8 and labeled proteins were analyzed by polyacrylamide gel(12.5%) electrophoresis. A fluorograph is shown.

FIG. 9 relates to dissociation of the purified gp80 into gp36. HIV-2infected CEM cells were labeled (17 hr) with [³⁵S] methionine and thevirus pellets were suspended is lysis buffer containing Triton. Thesevirus extracts (material corresponding from 2×10⁷ cells) wereimmunoprecipitated using mAb 1H8, and gp80 was purified by preparativegel electrophoresis. Equal aliquots of the purified gp80 preparationwere lyophilized and suspended in 100 mM acetate at pH 6.8, 5.8, and 4.8containing 1% (v/v) SDS, 100 units/ml aprotinin and 5 mM EGTA (toinhibit calcium-dependent proteolysis). All the samples were incubatedat 30° C. for 50 min before dilution in 2 fold concentratedelectrophoresis buffer. Samples were analyzed by polyacrylamide gel(12.5%) electrophoresis. A fluorograph is shown.

FIG. 10 substantiates that the transmembrane glycoprotein of SIV existsas a dimer. Section Cell: SIV-mac infected HUT-78 cells and HIV-2infected CEM cells were labeled for 16 hr with [³H] glucosamine (200μCi/ml; 4×10⁶ cells/ml). Extracts (prepared in lysis buffer containingTriton) from infected cells were purified by immunoprecipitation usingmAb 1H8 and the labeled proteins were analyzed by polyacrylamide gel(12.5%) electrophoresis. A fluorograph is shown.

FIG. 11 is a schematic pathway of HIV-2 envelope glycoproteinprocessing.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As a result of this invention, the processing of HIV-2 envelopeglycoproteins has now been characterized. An 80-Mr glycoprotein (gp80)was produced in HIV-2 infected cells along with three otherglycoproteins that were recently reported: the extracellularglycoprotein (gp125), the envelope glycoprotein precursor (gp140), andthe transient dimeric form of gp140 (gp300).

The gp125 and gp80 were detectable after the synthesis of gp14 and theformation of gp300. Among these four glycoproteins, only gp80 and gp125were associated with HIV-2 virions. As the other glycoproteins, gp80 wasrecognized by all HIV-2 positive sera. A murine polyclonal antibodyraised against purified gp300 recognized all four glycoproteins. On theother hand, a monoclonal antibody specific for transmembraneglycoprotein of HIV-2 recognized gp140, gp300, and gp80, thus indicatingthat gp80 should be related to the transmembrane pro tein of theenvelope. Heating (95° C., 5 min) of cellular or viral extracts in 1%SDS resulted in the dissociation of gp80 into the monomer gp36. Theseresults suggest that during the processing of the HIV-2 envelopeglycoprotein, the dimeric form of the precursor becomes cleaved by thecellular protease to give the extracellular glycoprotein gp125 and thetransmembrane glycoprotein dimer gp80.

Dimerization of envelope glycoprotein precursor and the transmembraneglycoprotein was also observed in cells infected with simianimmunodeficiency virus (SIV), a virus closely related to HIV-2.Dimerization of the envelope precursor might be required for itsprocessing to give the mature envelope proteins, whereas thetransmembrane dimer might be essential for optimal structure of thevirion.

The results obtained in practicing this invention will now be describedin greater detail.

I. Detection of a 80 kDa Protein in HIV-2 Infected Cells and in theVirion

Recently, we reported that the precursor of HIV-2 envelope glycoproteinsis a 140-kDa protein (gp140), which requires the formation of ahomologous dimer during its processing into the mature products, theextracellular (gp125) and transmembrane (gp36) glycoproteins (Rey etal., 1989). In these studies, however, the level of gp36 was found to bevery low and in some experiments it was not detectable. It has now beendiscovered that, in fact, that is the case because the transmembranegiycoprotein exists as a homodimer with an electrophoretic mobility inpolyacrylamide gels at a position corresponding to a 80-kDa protein(FIGS. 1 to 6). Accordingly for convenience, this 80-kDa protein will bereferred to as gp80.

Crude extracts from uninfected or HIV-1_(BRU) and HIV-2_(ROD) infectedCEM cells were analyzed by an electrophoretic transfer immunoblottingassay (Western blot) using an HIV-1 positive serum and three differentHIV-2 positive sera from AIDS patients (FIG. 1). The HIV-2 specific seraidentified the envelope precursors (gp140 and gp300) and in additionrecognized strongly the 80-kDa protein (gp80). These sera were specificfor HIV-2 proteins since they did not recognize HIV-1 proteins whichwere detectable using HIV-1 specific serum: the envelope glycoproteinprecursor (gp160) and gaq precursors (p55 and p40). The relation of gp80to HIV-2 infection was demonstrated by several results in which gp80 wasnot identified by HIV-1 positive serum nor was it found in HIV-1infected cells.

Western blot analysis of viral pellets prepared by centrifugation(100,000 g for 30 min) of infected culture medium indicated that gp80was also detectable in HIV-2 particles with the extracellularglycoprotein, gp125 (see below).

II. Synthesis of gp80 in HIV-2 Infected Cells

Preliminary experiments indicated that all HIV-2 positive sera canimmunoprecipitate gp80 in addition to the envelope precursors gp140 andgp300 and the extracellular glycoprotein, gp125. In order tocharacterize the synthesis of gp80, an HIV-2 positive serum whichrecognizes mainly the envelope proteins was used. Purified antibodiesfrom this serum were coupled to CNBr activated Sepharose (HIV-2serum-Sepharose) and was used as an immunoaffinity column to purifyenvelope glycoproteins. HIV-2 infected cells were labeled with [³H]glucosamine, and at different times (2, 3, 4, 6, and 8 hr) extracts wereprepared from infected cells as well as from virus pellets. All sampleswere purified on HIV-2 serum-Sepharose and labeled proteins wereanalyzed by polyacrylamide gel electrophoresis (FIG. 2). At 2 hr, gp140and gp300 were the only labeled proteins detectable in infected cells;gp125 and gp80 became detectable 3 to 4 hr after the start of thelabeling during which time they became also detectable in virus pelletsprepared from the culture medium. At 6 to 8 hours after the start oflabeling, gp125 and gp80 became clearly detectable. These resultsindicate, therefore, that gp80 is associated with virus particles andsuggest that gp80 might be a mature product of a precursor whichrequires processing. In these experiment, we could also detect some[3_(H)] glucosamine labeled gp36, but only intracellularly. The identityof the labeled 200 kDa protein is not known (FIG. 2). It is probably acellular protein since it was not immunoprecipitated by other HIV-2positive sera.

These kinetics results for the synthesis of HIV-2 envelope giycoproteinsare in accord with previous results. In HIV-2 infected cells, gp140 isthe first envelope product detectable at 15 min after a pulse-labeling.During a period of chase, the dimeric form of the envelope precursor(gp300) becomes detectable at 0.5 hr, whereas the mature extracellularglycoprotein (gp125) becomes detectable at 1.5 to 3 hr (Rey et al.,1989).

III. Identification of gp80 by Polyclonal Antibodies Against HIV-2Envelope Precursor

In order to characterize HIV-2 envelope glycoproteins, polyclonalantibodies against the purified dimeric precursor, gp300, were prepared.For this purpose, gp300 was first partially purified by animmunoadsorbent with antibodies from HIV-2 seropositive patient serumbefore purification by preparative electrophoresis. Five mice wereimmunized with 5 μg of this purified gp300 preparation administeredintraperitoneally five times at 10 days interval. Poly(A)·poly(U) (200μg) were used as an adjuvant which was administered mixed with theantigen (Materials and Methods), infra (see page 36). All mice developedantibodies against gp300. These antibodies are referred to as anti-gp300polyclonal antibodies.

FIG. 3 shows a Western blot analysis using antibodies from one of theimmunized mice. Anti-gp300 antibodies reacted specifically with gp300,but also with gp140, gp125, and gp80 present in HIV-2 infected cells. Nospecific signal was observed in uninfected or HIV-1 infected CEM cells.The labeling of a 60 kDa protein with anti-gp300 antibodies was probablynot specific since it was observed in cell extracts irrespective ofvirus infection (FIG. 3, section Cells) and in some experiments it wasnot at all observed.

These polyclonal antibodies were also used in a similar Western blotassay using extracts from HIV-2 infected cells as well as from the viruspellet. In the cellular extracts, the antibodies recognized gp300,gp140, and gp80 (FIG. 3, section HIV-2 lane C). In the viral extracts,they recognized gp125, gp80, and a 36 kDa protein which is probably thetransmembrane glycoprotein, gp36 (FIG. 3, section HIV-2 lane V). Onprolonged exposures, it was also possible to see a signal at theposition of gp36 in cellular extracts (data not shown). It isinteresting here to note that the level of gp80 and gp36 was much higherin the viral pellet compared to the cellular extract.

These results indicate that polyclonal antibodies raised against theenvelope precursor identify gp80 along with all the components of HIV-2envelope. Thus, gp80 should be related to HIV-2 envelope. The fact thatgp80 is associated with the virus suggests that it is a mature product.

IV. Identification of gp80 by the Monoclonal Antibody 1H8 Specific forthe Transmembrane Glycoprotein of HIV-2

The monoclonal antibody (mAb 1H8) was used in a Western blot assay todetermine whether viral proteins can be identified. In HIV-2 infectedcells, mAb 1H8 identified gp300, gp140, and gp80, whereas in the HIV-2pellet it identified mainly gp80 and weakly gp36 and gp300 (FIG. 4). Thepresence of low levels of gp300 in the virus pellet was probably due tosome contamination from lysed cells, since it is a cellular protein (Reyet al., 1989). The weak signal with gp36 probably reflects low levels ofthis protein. The mAb 1H8 did not recognize proteins in extracts fromHIV-1 infected cells or from the virus pellet. Furthermore, it did notrecognize the extracellular glycoprotein gp125 (FIG. 4). These resultsillustrate, therefore, the specificity of mAb 1H8 for HIV-2 envelopeprecursors (gp140 and gp300) and the transmembrane glycoprotein (gp36).The reactivity of mAb 1H8 was mapped to the amino acid sequence 579-604within the HIV-2 transmembrane glycoprotein using a synthetic peptidereferred to as p39′.

To show the specificity of mAb 1H8 reactivity with gp80, a western blotassay using extracts from HIV-2 virus pellet was carried out. After thetransfer of proteins, nitrocellulose sheets were incubated with mAb 1H8or anti-gp300 polyclonal antibodies in the absence or presence of 1μg/ml of peptide p39′ (FIG. 5). The mAb 1H8 gave a strong signal forgp80. In addition, a signal for gp36 was observed, but only after aprolonged exposure of the autoradiogram. Anti-gp300 polyclonalantibodies reacted with gp125, gp80, and gp36; the 60-kDa signal was notspecific (as in FIG. 3). Addition of peptide p39′ completely abolishedthe signals obtained with mAb 1H8, but not those obtained withanti-gp300 antibodies (FIG. 5). These observations confirm that thereactivity of mAb 1H8 should be with the 26 amino acid residuecorresponding to the amino acids 579 to 604 in the transmembraneglycoprotein of HIV-2. Consequently, a seauence corresponding to that ofpeptide p39′ should be present in gp80. The reactivity of anti-gp300antibodies was not modified by peptide p39′. Therefore, these antibodiesshould interact with other epitopes than that corresponding to peptide39′.

V. Immunoprecipitation of gp80 by Anti-gp300 Polyclonal Antibodies andby mAb 1H8

Anti-gp300 polyclonal antibodies immunoprecipitate gp300, gp140, gp125,and gp80, whereas mAb 1H8 immunoprecipitate gp300, gp140, and gp80 (FIG.6). Two cellular proteins (60 and 45 kDa) were also associated with theimmune complex preparations using both polyclonal and monoclonalantibodies (FIG. 6, lanes 0 and 2 hr). The presence of these 60 and 45kDa proteins in the immune complex preparation was due to their bindingto protein A Sepharose. This latter was used in order to recover immunecomplexes formed with the different antibodies.

HIV-2 infected cells were pulse-labeled for 1 hr before a chase of 2 and4 hr. Extracts from labeled cells (time 0, 2, and 4 hr) and the viralpellet recovered at 4 hr of chase were analyzed by immunoprecipitationassay using anti-gp300 polyclonal antibodies and mAb 1H8 (FIG. 6). Withboth polyclonal and monoclonal antibodies, gp80 was not detectable atthe period of pulse-labeling. It became clearly apparent at 2 hr ofchase in infected cells. When the chase was prolonged to 4 hr, then[³⁵S] methionine labeled gp80 became undetectable in infected cells.Analysis of viral pellets produced at 4 hr, indicated that as theextracellular glycoprotein (gp125), gp80 was associated with the virusparticles (FIG. 6). These results suggest that gp80 is a product of theprocessing of HIV-2 envelope. The fact that gp80 was identified bymonoclonal antibodies specific for the HIV-2 transmembrane glycoproteinindicated that it might be a dimeric form of gp36 (confirmed by theresults shown in FIGS. 8 and 9). The detection of gp80 was notrestricted to infected CEM cells, since it was also detectable in HIV-2infected T4 lymphocytes (data not shown).

Comparison of the results obtained by Western blot analysis and theimmunoprecipitation assays showed that patient sera, anti-gp300polyclonal antibodies, and mAb 1H8 recognize the denatured forms of gp80and gp125 better than their native forms (FIGS. 1, 2, 3, 4 and 6).Native forms of these mature glycoproteins probably have conformationswhich mask the epitopes identified by the different antibodies.

VI. Incorporation of Glucosamine and Fucose in gp80

Extracts from HIV-2 infected cells metabolically labeled with [³H]glucosamine and [³H] fucose were immunoprecipitated using mAb 1H8 andanti-gp300 polyclonal antibodies. In accord with the previous results(FIGS. 3-6), anti-gp300 antibodies immunoprecipitated sugar labeledgp300, gp140, gp125, and gp80, whereas mAb 1H8 immunoprecipitated gp300,gp140, and gp80. All these proteins incorporated [³H] glucosamine,whereas incorporation of [³H] fucose mainly occurred in gp125 and gp80(FIG. 7a). In these experiments, the labeling of gp36 with [³H]glucosamine was observed faintly after a prolonged exposure of theautoradiogram (data not shown; similar to FIG. 1, Rey et al., 1989). Theglycoprotein gp80 also can incorporate [³H] mannose as is the case forgp300, gp140, and gp125 (data not shown). Incorporation of these labeledsugars in gp80 and in other glycoproteins was completely blocked bytunicamycin, an antibiotic which inhibits N-linked glycosylation ofproteins (data not shown).

Asparagine-linked oligosaccharides (containing N-acetylglucosamine,mannose, and glucose) of glycoproteins undergo extensive processingafter their attachment to nascent proteins (Kornfeld and Kornfeld,1985). Oligosaccharide chains become trimmed in the endoplasmicreticulum and in the Golgi apparatus before the transfer of fucose andsialic acid residues. Therefore, incorporation of fucose residues occurslate in the glycosylation pathway. Accordingly, in HIV-2 infected cells[³H] fucose becomes incorporated mainly in gp125 and gp80, two proteinswhich are mature products of the HIV-2 envelope precursor (see FIGS. 6and 7).

To confirm that gp80 was produced during the processing of envelopeprecursor, HIV-2 infected cells were labeled with [³⁵S] methionine inthe absence or presence of the glucosidase inhibitor, castanospermine(Saul et al., 1983). Culture supernatants were then assayed byimmunoprecipitation using an HIV-2 positive patient serum whichrecognizes several viral proteins. In the presence of castanospermine,production of gp80 and gp125 were markedly reduced, whereas theproduction of the major core protein (p26) was not significantlyaffected (FIG. 7b).

VII. Dissociation of gp80 into gp36

Preliminary experiments suggested that gp80 could be dissociated to givegp36. For this reason, experiments were carried out to optimizeconditions under which the dissociation of gp80 might occur, such as,high salt, acidic pH, ionic detergent, EDTA, and EGTA.

Previously, it was reported that the dimeric form of the envelopeprecursor (gp300) can be dissociated by incubation in slightly acidicbuffer (Rey et al., 1989). Although this latter method also works forgp80, but in a buffer less than pH 6, most of gp80 becomes degraded. Inall experiments, extracts were prepared from infected cells or fromviral pellets by a lysis buffer containing non-ionic detergent, TritonX-100. Under these conditions, gp30 and gp80 are not dissociated evenafter addition of the ionic-detergent SDS.

The effect of SDS was investigated when it is used instead of Triton forthe preparation of extracts. HIV-infected cells were labeled with [³⁵S]methionine before preparation of extracts by solubilization in lysisbuffer containing either 1% Triton X-100 or 1% SDS. These extracts werethen diluted 10 fold in lysis buffer without detergent andimmunoprecipitated using mAb 1H8. The immune-compiex preparations fromTriton-extracts showed the presence of [³⁵S] methionine-labeled bandscorresponding to gp300, gp140, gp80 and a faint-band of gp36 (FIG. 8,section C lane 1). On the other hand, when extracts were prepared withSDS, then gp300 and gp80 were almost undetectable whereas the level ofgp140 and gp36 was increased (FIG. 8, section C lane 2). Thus in thepresence of ionic detergent, the dimeric forms gp300 and gp80 weredissociated giving rise to gp140 and gp36, respectively. Dissociation ofthe purified [³⁵S] methionine labeled gp300 gives only gp140 (Rey etal., 1989). Accordingly, gp36 should arise from the dissociation ofgp80.

It should be noted that the degradation of proteins occurred also in thepresence of SDS since not all the label in gp300 and gp80 was recoveredin the dissociated proteins (FIG. 8, section C). The presence of 200units/ml aprotinin and 0.2 mM PMSF did not prevent such degradationduring incubation with SDS. It might be that the dimeric forms ofproteins have a conformation which can resist proteolysis. Dissociationof gp300 and gp80 might then lead to conformational modifications makingthe proteolytic sites accessible.

Dissociation experiments were also carried out with the HIV-2 pellet.[³⁵S] methionine labeled viral proteins were solubilized in lysis buffercontaining 1% Triton or 1% SDS and in RIPA buffer containing 0.1% SDSand 1% deoxycholate. Extracts in 1% SDS lysis buffer were also heated at95° C. All the extracts were then immunoprecipitated with mAb 1H8 andanalyzed by polyacrylamide gel electrophoresis (FIG. 8, section V). Inthe immune-complex prepared from Triton-extracts, gp80 and gp36 were themajor proteins immunoprecipitated by mAb 1H8. On the other hand, gp80became undetectable whereas the level of gp36 was slightly increased inSDS-extracts. The degree of degradation in SDS-extracts must have beendramatic since most of the label disappeared. A somewhat better resultwas observed with the RIPA buffer in which most of gp80 was dissociatedand about 30% of the label was recovered in gp36 (FIG. 8, section V,lane 4).

In order to show that gp80 is composed of only gp36, dissociationexperiments were carried out using [³⁵S] methionine labeled gp80purified by mAb 1H8 immunoprecipitation and by preparative gelelectrophoresis. Lyophilized gp80 preparations were then suspendeddirectly in an acetate buffer with SDS at pH 6.8, 5.8, and 4.8. At pH4.8, all gp80 was converted to gp36 (FIG. 9).

VIII. The Transmembrane Glycoprotein of SIV-mac Exists in a Dimeric Form

Previously, it was shown that the glycoprotein precursor (gp140) of SIVforms a dimer during its processing (Rey et al., 1989). For this reasonit was important to investigate whether the transmembrane glycoproteinwas also detectable as a dimer. SIV and HIV-2 infected cells werelabeled with [³H] glucosamine and extracts prepared by lysis buffercontaining Triton were immunoprecipitated with mAb 1H8. As in FIG. 7,the monoclonal antibody precipitated gp300, gp140, and gp80 from HIV-2infected cells. In addition, a very faint band corresponding to gp36 wasdetected (FIG. 10). In SIV infected cells, the monoclonal antibodyprecipitated three glycosylated proteins: the envelope precursor gp140,the dimer precursor gp300, and a 65 kDa protein (gp65) which wasprobably the counterpart of HIV-2 gp80 (FIG. 10). As gp80, gp65 wasfound to be associated with SIV virus particles.

It should be noted that in these experiments, monomeric forms of thetransmembrane glycoprotein of HIV-2 ROD and SIV-mac were not detectable.The HIV-2 ROD amino-acid sequence 579-604 corresponds to SIV-macsequence 595-620 (5,16). Since these two sequences are highlyhomologous, then mAb 1H8 cross-reacts with envelope proteins of bothHIV-2 ROD and SIV-mac.

By the use of a monoclonal antibody, Veronese et al. (33) have recentlyreported that the transmembrane glycoprotein of SIV-mac is a 32 kDAprotein (gp32). However, in their immunoprecipitation assays, theyreported the presence of unidentified 75 and 300 kDa proteins at highlevels along with the envelope precursor gp140. In analogy with the dataherein, the 75 kDa protein is probably the dimeric form of thetransmembrane glycoprotein gp32 (FIG. 10), whereas the 300 kDa proteinshould be the dimeric form of the envelope precursor previously reported(29).

This invention thus elucidates the processing of HIV-2 envelopeglycoprotein. The unusual feature of this processing is that theenvelope precursor requires the formation of a homologous dimer in orderto become transported and processed through the Golgi apparatus (Rey etal., 1989). The precursor gp140 becomes dimerized in the roughendoplasmic reticulum and the resulting gp300 dimer intermediaryprecursor is then transported to the Golgi apparatus where it is furtherprocessed. Finally, the dimer is transported to the plasma membrane andcleaved by the cellular protease to yield the mature HIV-2 envelopeglycoproteins: the extracellular glycoproteins (gp125) in monomericforms and the transmembrane glycoproteins (gp36) in dimeric forms (gp80)(FIG. 11).

Referring to FIG. 11, the envelope polypeptide during its synthesisbecomes glycosylated to give rise to the “hypothetical glycoproteinprecursor” gp150 which becomes rapidly trimmed by the rough endoplasmicreticulum (RER) glucosidases I and II and mannosidase to give rise togp140. This monomer becomes dimerized and the resulting gp300 “dimerintermediary precursor” is then transported to the Golgi apparatus.Further trimming of gp300 is carried out by the Golgi mannosidasesbefore transfer of fucose and sialic acid residues in the medial andtrans Golgi. Finally, the dimer is cleaved by the cellular protease toyield the extracellular envelope glycoprotein (ECG) gp125 and thedimeric form of the transmembrane glycoprotein (TMG), gp80.

In a previous study (Rey et al., 1989), it was suggested that theprocessed gp300 becomes dissociated before transport to the plasmamembrane. This is probably not the case, because now there is evidenceto indicate that the transmembrane proteins become produced ashomodimers. In the previous study, monensin was used as an inhibitor ofthe transport of membrane glycoproteins and secretory proteins from theGolgi apparatus (Johnson and Schlesinger, 1980). In the presence ofmonensin, a 135-KDa protein (gp135), which might have been thedissociated product of gp300, was accumulated in HIV-2 infected cells.This production of gp135 was probably an artifact triggered by monensindue to accumulation of the processed gp300 in the Golgi apparatus.

The mechanism of dimerization of the envelope glycoprotein precursor isnot yet clear. This is an intrinsic property of the polypeptide moietyof the envelope precursor. The fact that the transmembrane glycoproteinsexist in dimeric forms (gp80), suggests that dimerization of gp140occurs through interactions between the transmembrane regions of theenvelope precursor. Dissociation of the dimeric forms (gp300 and gp80)might occur at slightly acidic pH. Accordingly, dimerization of gp140might be pH dependent and occurs in a compartment in the roughendoplasmic reticulum which favors the fusion of two gp140 precursormolecules.

Several observations indicate that formation of gp300 is not an artifactobserved in HIV-2 infected CEM cells: (1) Pulse-chase experiments inHIV-2 infected CEM cells indicate that gp140 is first synthesized beforethe formation of gp300 which is itself detected few hours before theproduction of mature envelope proteins, gp125 and gp80; (2) A similarkinetics for the detection of gp140, gp300, gp125, and gp80 is alsoobserved in T4 lymphocytes infected with HIV-2; (3) The fate of gp140 isthe formation of gp300. This latter is well illustrated by experimentsin which transport of the dimer from the endoplasmic reticulum to theGolgi apparatus is blocked by glucosidase inhibitors. In the presence ofthese inhibitors, all monomer precursors synthesized by pulse-labelingof infected cells become dimerized during the period of chase.Dimerization of gp140 is also not a consequence of experimentalconditions in our studies. Pulse-chase experiments carried out atreduced temperatures (15-20° C.) to block transport in the endoplasmicreticulum, show that the monomer precursors synthesized bypulse-labeling of infected cells do not form dimers when the chase iscarried out at reduced temperatures. Thus, these results indicate thatformation of gp300 requires transport of gp140 in a compartment with anenvironment favoring the process of dimerization. All these observations(Rey et al., 1989) emphasize that dimerization is a natural step in theprocessing pathway of gp140, i.e., dimerization is not due toaccumulation of unprocessed gp140, nor is it an artifact of theexperimental procedure.

The molecular weight of the dimeric form of the envelope precursor hadbeen estimated by polyacrylamide gel electrophoresis under denaturingconditions (Rey et al., 1989). In a 5% polyacrylamide gel containing0.1% bisacrylamide instead of 0.2% (wt/vol), this dimeric precursormigrated at a position corresponding to a 280-kDa protein (data notshown). In order to confirm the molecular weight of this dimer undernative conditions, gel filtration experiments were carried out using anS-300 Sephacryl column and [³⁵S] methionine labeled extracts from HIV-2infected cells prepared by lysis buffer containing Triton. Under theseexperimental conditions, gp300 eluted as the second peak after the peakof aggregated proteins. The transmembrane glycoprotein dimer eluted as a75-80 kDA protein after the peak of gp125 and before the peak ofbovine-serum-albumin (68 kDa) marker (data not shown). Theseobservations indicate that the molecular weight estimations of thenative and denatured dimers gp300 and gp80 are comparable. In vitro, thedissociation of these dimeric forms occurred in acidic pH and also inthe presence of the ionic-detergent SDS. When extracts were prepared inthe lysis buffer containing Triton, then the dimeric forms gp300 andgp80 resisted dissociation by SDS. The non-ionic detergent Triton,therefore, conserved the native forms of the envelope dimers, gp300 andgp80.

Dimeric forms of the envelope precursor and the transmembraneglycoprotein were also observed in cells infected with SIV but not withHIV-1. In the case of HIV-1, it has been reported that a smallproportion of the transmembrane glycoprotein in the HIV-1 particlesmight exist as a dimer linked by disulfide bonds (Bharat Parekh andRoger Walker, personal communication). The transmembrane dimers of HIV-2(gp80) and SIV (gp65), however, resist dissociation by reducing agents.Accordingly, dimerization of the envelope glycoprotein precursor can beconsidered a specific property of HIV-2 and SIV envelope geneexpression. This property could be used for characterization of new HIVisolates, as a convenient marker to describe their relationship to HIV-1or HIV-2. Dimerization of the envelope precursor might be required forthe processing of the envelope precursor to yield the mature envelopeglycoproteins. The dimeric form of the transmembrane glycoprotein mightbe essential for optimal structure of the virion and thus its capacityto fuse with the cellular membrane and be infectious. In vitrodissociation of the transmembrane dimer leads to a dramatic degradation.It might be, therefore, that the dimeric forms of this glycoprotein havea conformation which resists proteolysis.

Previously, the formation of oligomeric complexes of some viralstructural glycoproteins has been reported. For example, thehemagglutinin (HA) of influenza virus exists as a trimer which could bestabilized by cross-linking agents (5, 36, 37). The G glycoprotein ofthe vesicular stomatitis virus (VSV) also forms a trimer (11, 24). Morerecently, the envelope glycoprotein precursor of the Rous sarcoma viruswas reported to form a trimer held by a disulfide linkage (12). In allthese studies, formation of oligomeric complexes has been associatedwith their intracellular transport from the endoplasmic reticulum. Inthe case of the RSV the trimeric structure of the transmembraneglycoprotein seems to be the functional form found in virions.Therefore, the results observed in the RSV are analogous with thosepresented here, i.e., oligomerization of the HIV-2 and SIV envelopeglycoprotein precursor and the transmembrane glycoprotein. In contrastto the RSV however, the dimeric forms of the HIV-2 and SIV are stableand are not dissociated by reducing agents. Nevertheless, all theseobservations enforce the suggestion that efficient processing of someglycoproteins requires tne formation of oligomeric structures, and insome cases oligomeric forms of the mature glycoprotein might beessential for infectivity. Accordingly, antiviral agents designed toblock the formation of oligomeric precursors or cause dissociation ofoligomeric complexes of the mature proteins, might be employed toprevent virus replication and its spreading.

Following is a more detailed description of the experimental proceduresused in this invention.

Materials

L-[³⁵S] Methionine (specific activity >1000 μCi/mmol, L-[6-³H] Fucose(specific activity: 45-70 μCi/mmol), D-[6-³H] Glucosamine (specificactivity: 20-40 μCi/mmol) were purchased from Amersham (Amersham, UK).Castanospermine and tunicamycin were obtained from Boehringer-Mannheim(Manneheim, West Germany). Poly(A)·poly(U) was the generous gift of M.Michelson, Institut Curie, Paris, and is prepared according toHovanessian et al. (J. Interferon Res. 1982, 2:209-216).

Virus and Cells

HIV-1_(BRU) isolate of the human immunodeficiency virus type 1(Montagnier et al., 1984), HIV-2_(ROD) isolate of the humanimmunodeficiency virus type 2 (Clavel et al., 1986), and Simianimmunodeficiency virus, SIVmac₁₄₂ (Daniel et al., 1985) were used inthis study.

The different cell lines and human lymphocytes were cultured insuspension medium RPMI-1640 (GIBCO-BRL, Cergy-Pontoise France)containing 10% (v/v) fetal calf serum; 2 μg/ml Polybrene (Sigma) wasadded for HIV infected cell cultures. CEM clone 13 cells are derivedfrom the human lymphoid cell line CEM (ATCC-CCL119) and express the T4antigen to a high level. Five days after infection with HIV-1_(BRU) orHIV-2_(ROD) isolates, about 80-90% of the cells produce viral particlesand can be identified by a cytopathic effect corresponding tovacuolisation of cells and appearance of small syncitia. The HUT-78 cellline is another human T4 positive lymphoid cell line (Gazdar et al.,1980) that is highly permissive for the replication of SIVmac₁₄₂ (Danielet al., 1985). Peripheral blood lymphocytes from healthy blood donorswere stimulated for three days with 0.2% (w/v) phytohemagglutininfraction P (Difco, Detroit, USA) in RPMI-1640 medium supplemented with10% fetal calf serum. Cells were then cultured in RPMI-1640 mediumcontaining 10% (v/v) T cell growth factor (TCGF, Biotest). Afterinfection with HIV-2. lymphocytes were cultured in presence of 10% (v/v)TCGF and 2 μg/ml Polybrene.

Metabolic Labeling of Cells

For metabolic labeling of proteins, infected cells were incubated for 16hours at 37° C. in MEM culture without L-methionine and serum, butsupplemented with 200 μCi/ml [³⁵S] methionine. For metabolic labeling ofglycoproteins, infected cells were in cubated for 16 hours at 37° C. inMEM culture medium lacking serum and glucose but supplemented with 200μCi/ml ³H-Fucose or 200 μCi/ml ³H-glucosamine.

Cells and Viral Extracts

Cell pellets corresponding to 10⁷ cells were resuspended in 100 μl ofbuffer: 10 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.2 mM PMSF, 100units/ml aprotinin (Iniprol, Choay) before addition of 100 μl of thesame buffer containing 2% (v/v) Triton X-100. Cell extracts werecentrifuged at 12,000 g for 10 minutes, and the supernatant was storedat −80° C. until used. For viral extract preparations, 100 μl of 10Xlysis buffer (100 mM Tris-HCl pH 7.6, 1.5M NaCl, 10 mM EDTA, 10% (v/v)Triton X-100, 100 units/ml aprotinin) were added per ml of clarifiedsupernatant from infected CEM cells and processed as above. For thepreparation of extracts from virus pellets, culture medium from infectedcells was first centrifuged at 12,000 g for 10 minutes before high speedcentrifugation at 100,000 g for 30 to 60 min. Virus pellets (materialfrom 10⁷ cells) were then solubilized in 200 μl of lysis buffer.

Preparation of an Immunoadsorbant with Antibodies from an HIV-2Seropositive Patient

Immunoglobulins from the serum of an HIV-2 seropositive patient wereprecipitated with 50% (NH₄)₂SO₄, dissolved in 20 mM sodium phosphate (pH8.0) and further purified on a DEAE cellulose column (DE52, Whatman) byelution with 20 mM sodiam phosphate (pH 8.0). Immunogiobulins purifiedin this manner were judged to be 90% pure. The antibodies weresubsequently coupled to CNBr-activated Sepharose CL 4B according to atechnique described (Berg, 1977). Two milligrams of IgG were coupled perml of Sepharose CL 4B. This immunoadsorbant is referred to as HIV-2serum-Sepharose.

Preparation of HIV-2 Proteins on an Immunoaffinity Column

Cell extracts from HIV-2 producing CEM cells were first diluted in twovolumes of binding buffer (20 mM Tris-HCl pH 7.6, 50 mM KCl, 150 mMNaCl, 1 mM EDTA, 1% (v/v) glycerol, 7 mM β-mercaptoethanol, 0.2 mM PMSF,100 units/ml aprotinin) before incubation with one volume of HIV-2serum-Sepharose. Supernatants from HIV-2 producing cells were processedas cell extracts except that only one tenth of binding bufferconcentrate 10X was added per volume of supernatant. The binding wascarried out overnight, then the column was washed batchwise in bindingbuffer. Proteins bound to the column were eluted by boiling inelectrophoresis sample buffer (125 mM Tris-HCl pH 6.8, 1% (w/v) SDS, 2Murea, 20% glycerol, 0.5% β-mercaptoethanol). Eluted proteins wereresolved by electrophoresis on 7.5% polyacrylamide-SDS gels containing6M urea and 0.1% bisacrylamide instead of 0.2% (w/v).

Preparative Electrophoresis

HIV-2 glycoproteins (gp300 or gp80) eluted from the affinity column wereresolved by polyacrylamide gel electrophoresis as previously described,and the regions of the gel containing the viral glycoproteins were cutout by reference to the position of prestained molecular weight proteinmarkers (BRL). Glycoprotein gp300 was eluted by incubation for 16 hoursat 4° C. in elution buffer (0.1M NaHCO₃, 0.5 mM EDTA, 0.05% (w/v) SDS,0.2 mM PMSF). The glycoprotein fractions thus obtained were lyophilizedand kept refrigerated until used. Glycoprotein gp80 was electroeluted inbuffer containing 4 mM Tris-HCl pH 7.6, 2 mM sodium acetate, and 2 mMEDTA.

Preparation of Murine Polyclonal Antibodies, Anti-gp300

HIV-2 envelope glycoprotein gp300 was purified from extracts of infectedCEM cells (3×10⁸ cells) by immunoaffinity chromatography on the HIV-2serum-Sepharose and followed by preparative gel electrophoresis (Rey etal., 1989). The purified preparation of gp300 was dissolved in 10 ml of150 mM NaCl containing 0.5M urea and 1 mg/ml of mouse serum proteins anddialyzed for 24 hours against the solution containing 150 mM NaCl and0.5M urea. The dialysate was then centrifuged and 2 ml aliquots werestored at −80° C.

Five mice (8 weeks old) were injected intraperitoneally, five times at12 days interval with 350 μl of the gp300 preparation (about 0.1 μg ofgp300). Poly(A)·poly(U) (200 μg; 1 mg/ml in 150 mM NaCl) was used as anadjuvant which was administered intravenously during each immunization(Hovanessian et al., 1988). Five days before the last injection, micewere injected intraperitoneally with a suspension of 10⁶ sarcoma 180/TGcells to prepare hyperimmune ascitic fluid (Hovanessian et al., 1988). Aweek following the booster, mice were sacrificed and the ascitic fluidswere collected. Ascitic cells were removed by centrifugation (200 g, 5min) and the peritoneal fluid was collected.

Production and Characterization of Monoclonal, mAb 1H8

HIV-2 ROD virions were cultivated in CEM cells and purified fromconcentrated culture supernatants by banding in sucrose gradients.Purified virus was disrupted in 0.5% Triton X-100, 150 nM NaCl, 50 mMTris, pH 8.0, 1% aprotinin (Sigma) and clarified byultra-centrifugation. The viral extract was then passed over aLentil-Lectin Sepharose 4B affinity column (Pharmacia), the columnwashed, and the bound glycoproteins eluted with 0.5 M methyla-D-monnopyranoside (Sigma), and dialyzed overnight againstphosphate-buffered saline. BALB-C mice were immununizedintraperitoneally with 0.3 mls of purified glycoproteins (2-5 μg)reattached to Lentil-Lectin Sepharose 4B (50-100 μl). The mice wereboosted every 4-6 weeks for 24 weeks with the same immunogen andmonitored for HIV-2 glycoprotein antibodies qualitatively by RIPA of[³⁵S] methionine labeled HIV-2 virion extracts and quantitatively on theGenetic Systems HIV-2 disrupted virion EIA. Three days after the lastinjection, spleen cells were fused with NSI myeloma cells according tothe method of Kohler and Milstein (22). 96-well fusion plates werescreened by hybridomas secreting anti-HIV-2 antibodies using the GeneticSystems HIV-2 disrupted virion EIA. Methods for the propagation andstabilization of cloned hybridomas and for ascites production have beenpreviously described (16). Hybridoma culture supernatants were screenedby RIPA and Western blot analysis. Monoclonal antibody (mAb) 1H8, whichreacted with the transmembrane glycoprotein, was further mapped toamino-acid squence 579-604 within the HIV-2 transmembrane glycoproteinusing a synthetic peptide based EIA (32). The HIV-2 amino-acid sequence579-604 is highly conserved among all HIV-2 and SIV isolates thus farsequenced accordingly, monoclonal antibody 1H8 cross-reacts with HIV-2and SIV isolates thus far tested. The synthetic peptide p39′ wassynthesized according to the amino-acid sequence 579-604 deduced fromthe nucieotide sequence of the HIV-2 ROD envelope. The amino-acidsequence of peptide p39′ is the following

VTAIEKYLQDQARLNSWGCAFRQVCH.

Radio-immunoprecipitation Assay (RIPA)

Cell or viral extracts (20 μl) (material corresponding to 1×10⁶ infectedcells) were first diluted in two volumes of RIPA buffer [(10 mM Tris-HClpH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (v/v), 0.2% sodiumdeoxycholate (wt/v), 0.1% SDS (wt/v), 7 mM 2 β-mercaptoethanol, 0.2 mMPMSF, 100 units/ml of aprotinin (Iniprol, Choay)]. Diluted extracts werethen incubated (45 min, 4° C.) with polyclonal or monoclonal antibodies(2-5 μl). Protein A-Sepharose was then added and the samples werefurther incubated for 3 hr at 4° C. These samples were washed in theRIPA buffer. Proteins recovered by immunoprecipitation were eluted byheating (95° C., 5 min) in the electrophoresis sample buffer [125 mMTris-HCl, pH 6.8, 1% SDS (wt/v), 20% glycerol (v/v), 1% 2β-mercaptoethanol]. Eluted proteins were resolved by electrophoresis in7.5-12.5% polyacrylamide SDS gels containing 0.1% bisacrylamide insteadof 0.2% (wt/v).

Electrophoretic Transfer Immunoblot Analysis: Western Blot

Proteins were subjected to analysis by polyacrylamide gelelectrophoresis before being electrophoretically transferred to 0.45 μmnitrocellulose sheets (Schleicher and Schull, Dassel, FRG) in electrodebuffer (20 mM Tris base, 150 mM glycine, 20% methanol, v/v) as described(Burnette 1981). The electrophoretic blots were saturated with 5% (w/v)non-fat dry milk in PBS (Johnson et al., 1984). They were then incubatedin a sealed bag (overnight 4° C.) either with HIV-1 or HIV-2 positivesera (at 1:100 dilution) or with mouse polyclonal or monoclonalantibodies (at 1:200 dilution) in PBS containing 10% FCS. The sheetswere subsequently washed in PBS, PBS containing 5% Nonidet P-40 and thenresaturated in PBS containing non-fat milk (5%). The washed sheets werethen incubated (2 hr, room temperature) in a sealed bag either with apreparation of ¹²⁵I-labeled protein A (Amersham, >30 mCi/mg) to revealthe human polyclonal antibodies in the HIV-1 or HIV-2 sera or with apreparation of ¹²⁵I-labeled goat anti-mouse immunoglobulins (Amersham;2-10 μCi/μg). The sheets were removed from the bags and washed again,dried and autoradiographed (Kodak RP Royal, X-Ray films) for 24-48 hr.

It will be understood that the present invention is intended toencompass the Previously described proteins and glycoproteins inpurified form, whether or not fully glycosylated, and whether obtainedusing the techniques described herein or other nethods. In a preferredembodiment of this invention, the retrovirus and polypeptides aresubstantially free of human tissue and human tissue components, nucleicacids, extraneous proteins and lipids, and adventitious microorganisms,such as bacteria and viruses. It will also be understood that theinvention encompasses equivalent proteins and glycoproteins havingsubstantially the same biological and immunogenic properties. Thus, thisinvention is intended to cover serotypic variants of the proteins andglycoproteins of the invention.

The proteins and glycoproteins of this invention can be obtained byculturing HIV-2 in susceptible mammalian cells of lymphocytic lineage,such as T-lymphocytes or pre-T-lymphocytes of human origin or non-humanprimate origin (e.g. chimpanzee, African green monkey, or macaques.) Anumber of different lymphocytes expressing the CD4 phenotypic marker canbe employed. Examples of suitable target cells for HIV-2 infection aremononuclear cells prepared from peripheral blood, bone marrow, and othertissues from patients and donors. Alternatively, established cell linescan be employed. For example, HIV-2 can be propagated on blood-donorlymphocyte cultures, followed by propagation on continuous cell strainsof leukemic origin, such as HUT 78. HUT 78 is a well characterizedmature human T cell line, which has been deposited at CollectionNationale Des Cultures De Micro-organismes (CNCM) at the PasteurInstitut in Paris, France on Feb. 6, 1986, under culture collectiondeposit accession number CNCM I-519. Another suitable target for HIV-2infection and production of the proteins and glycoproteins of theinvention is the T-cell line derived from an adult with lymphoidleukemia and termed HT. HT cells continuously produce virus afterparental cells are repeatedly exposed to concentrated cell culturefluids harvested from short-term culture T-cells grown in TCGF thatoriginated from patients with LAS or AIDS. In addition, there areseveral other T or pre-T human cell lines. such as CEM and MOLT 3 thatcan be infected and continue to produce HIV-2. Furthermore,B-lymphoblastic cell lines can also be productively infected by HIV.Montagnier et al, Science, 225:63-66(1984).

The proteins and glycoproteins of the invention can be produced in thetarget cells using the culture conditions previously described, as wellas other standard techniques. For instance, infected human lymphocytescan be stimulated for three days by phytohemaglutinin (PHA). Thelymphocytes can be cultured in RPMI-1640 medium to which has been added10% fetal calf serum, 10⁻⁵M beta mercaptoethanol, interleukin 2, andhuman alpha anti-interferon serum. Barre-Sinoussi et al, Science,220:868-871 (1983). In addition, techniques for the-propagation of HIV-2in HUT 78 and CEM cell lines are described in copending U.S. applicationSer. No. 835,228, filed Mar. 3, 1986, the entire disclosure of which isrelied upon and incorporated by reference herein.

The production of virus in the cell cultures can be monitored usingseveral different techniques. Supernatant fluids in the cell culturescan be monitored for viral reverse transcriptase activity. Electronmicroscopic observation of fixed and sectioned cells can also be used todetect virus. In addition, virus can be detected by transmitting thevirus to fresh normal human T-lymphocytes (e.g., umbilical cord blood,adult peripheral blood, or bone marrow leukocytes) or to establishedT-cell lines. Testing for antigen expression by indirectimmunofluorescence or Western Blot procedures using serum fromseropositive donors can also be employed. In addition, nucleic acidprobes can be utilized to detect viral production.

After a sufficient period of time for viral multiplication to takeplace, infected cells can be separated from the culture medium anddisrupted to expose intracellular proteins using conventionaltechniques. For example, physical shearing, homogenization, sonication,detergent solubilization, or freeze-thawing can be employed. The viralproteins released by these cells can be separated from the othercellular components and purified using standard biochemical procedures.For example, virus can be recovered by ultra centrifugation, and theviral proteins can then be solubilized by detergent and then purified bygel filtration, ion-exchange chromatography, affinity chromatography,dialysis, or by the use of monoclonal antibodies or by combinations ofthese procedures. A thorough purification of the antigens of theinvention can be performed by immunoreaction with the sera of patientsknown to possess antibodies effective against the antigens, withconcentrated antibody preparations such as polyclonal antibodies, orwith monoclonal antibodies directed against the antigens of theinvention.

The proteins and the glycoproteins of the present invention can be usedas antigens to identify antibodies to HIV-2 and SIV in materials and todetermine the concentration of the antibodies in those materials. Thus,the antigens can be used for qualitative or quantitative determinationof the retrovirus in a material. Such materials of course include humantissue and human cells, as well as biological fluids, such as human bodyfluids, including human sera. When used as a reagent in an immunoassayfor determining the presence or concentration of the antibodies toHIV-2, the antigens of the present invention provide an assay that isccnvenient, rapid, sensitive, and specific.

More particularly, the antigens of the invention can be employed for thedetection of HIV-2 by means of immunoassays that are well known for usein detecting or quantifying humoral components in fluids. Thus,antigen-antibody interactions can be directly observed or determined bysecondary reactions, such as precipitation or agglutination. Inaddition, immunoelectrophoresis techniques can also be employed. Forexample, the classic combination of electrophoresis in agar followed byreaction with anti-serum can be utilized, as well as two-dimensionalelectrophoresis, rocket electrophoresis, and immunelabeling ofpolyacrylamide gel patterns (Western Blot or immunoblot.) Otherimmunoassays in which the antigens of the present invention can beemployed include, but are not limited to, radioimmunoassay, competitiveimmunoprecipitation assay, enzyme immunoassay, and immunofluorescenceassay. It will be understood that turbidimetric, calorimetric, andnephelometric techniques can be employed. An immunoassay based onWestern Blot technique is preferred.

Immunoassays can be carried out by immobilizing one of theimmunoreagents, either an antigen of the invention or an antibody of theinvention to the antigen, on a carrier surface while retainingimmunoreactivity of the reagent. The reciprocal immunoreagent can beunlabeled or labeled in such a manner that immunoreactivity is alsoretained. These techniques are especially suitable for use in enzymeimmunoassays, such as enzyme linked immunosorbent assay (ELISA) andcompetitive inhibition enzyme immunoassay (CIEIA).

When either the antigen of the invention or antibody to the antigen isattached to a solid support, the support is usually a glass or plasticmaterial. Plastic materials molded in the form of plates, tubes, beads,or disks are preferred. Examples of suitable plastic materials arepolystyrene and polyvinyl chloride. If the immunoreaqent does notreadily bind to the solid support, a carrier material can be interposedbetween the reagent and the support. Examples of suitable carriermaterials are proteins, such as bovine serum albumin, or chemicalreagents, such as gluteraldehyde or urea. Coating of the solid phase canbe carried out using conventional techniques.

Depending on the use to be made of the proteins and glycoproteins of theinvention, it may be desirable to label them. Examples of suitablelabels are radioactive labels, enzymatic labels, fluorescent labels,chemiluminescent labels, and chromophores. The methods for labelingproteins and glycoproteins of the invention do not differ in essencefrom those widely used for labeling immunoglobulin. The need to labelnay be avoided by using labeled antibody to the antigen of the inventionor anti-immunoglobulin to the antibodies to tne antigen as an indirectmarker.

Once the proteins and glycoproteins of the invention have been obtained,they can be used to produce polyclonal and monoclonal antibodiesreactive therewith. Thus, a protein or glycoprotein of the invention canbe used to immunize an animal host by techniques known in the art. Suchtechniques usually involve inoculation, but they may involve other modesof administration. A sufficient amount of the protein or theglycoprotein is administered to create an immunogenic response in theanimal host. Any host that produces antibodies to the antigen of theinvention can be used. Once the animal has been immunized and sufficienttime has passed for it to begin producing antibodies to the antigen,polyclonal antibodies can be recovered. The general method comprisesremoving blood from the animal and separating the serum from the blood.The serum, which contains antibodies to the antigen, can be used as anantiserum to the antigen. Alternatively, the antibodies can be recoveredfrom the serum. Affinity purification is a preferred technique forrecovering purified polyclonal antibodies to the antigen, from theserum.

Monoclonal antibodies to the antigens of the invention can also beprepared. One method for producing monoclonal antibodies reactive withthe antigens comprises the steps of immunizing a host with the antigen;recovering antibody-producing cells from the spleen of the host; fusingthe antibody-producing cells with myeloma cells deficient in the enzymehypoxanthine-guanine phosphoribosyl transferase to form hybridomas;selecting at least one of the hybridomas by growth in a mediumcomprising hypoxanthine, aminopterin, and thymidine; identifying atleast one of the hybridomas that produces an antibody to the antigen;culturing the identified hybridoma to produce antibody in a recoverablequantity; and recovering the antibodies produced by the culturedhybridoma.

These polyclonal or monoclonal antibodies can be used in a variety ofapplications. Among these is the neutralization of correspondingproteins. They can also be used to detect viral antigens in biologicalpreparations or in purifying corresponding proteins, glycoproteins, ormixtures thereof, for example when used in affinity chromatographiccolumns.

The invention provides immunogenic proteins and glycoproteins, and moreparticularly, protective polypeptides for use in the preparation ofvaccine compositions against HIV-2. These polypeptides can thus beemployed as viral vaccines by administering the polypeptides to a mammalsusceptible to HIV-2 infection. Conventional modes of administration canbe employed. For example, administration can be carried out by oral,respiratory, or parenteral routes. Intradermal, subcutaneous, andintramuscular routes of administration are preferred when the vaccine isadministered parenterally.

The ability of the proteins, glycoproteins, and vaccines of theinvention to induce protective levels of neutralizing antibody in a hostcan be enhanced by emulsification with an adjuvant, ncorporation in aliposome, coupling to a suitable carrier, or by combinations of thesetechniques. For example, the proteins and glycoproteins of the inventioncan be administered with a conventional adjuvant, such as aluminumphosphate and aluminum hydroxide gel, in an amount sufficient topotentiate humoral or cell-mediated immune response in the host.Similarly, the polypeptides can be bound to lipid membranes orincorporated in lipid membranes to form liposomes. The use ofnonpyrogenic lipids free of nucleic acids and other extraneous mattercan be employed for this purpose.

The immunization schedule will depend upon several factors, such as thesusceptibility of the host to infection and the age of the host. Asingle dose of the vaccine of the invention can be administered to thehost or a primary course of immunization can be followed in whichseveral doses at intervals of time are administered. Subsequent dosesused as boosters can be administered as needed following the primarycourse.

The proteins and vaccines of the invention can be administered to thehost in an amount sufficient to prevent or inhibit HIV-2 infection orreplication in vivo. In any event, the amount administered should be atleast sufficient to protect the host against substantialimmunosuppression, even though HIV infection may not be entirelyprevented. An immunogenic response can be obtained by administering theproteins or glycoproteins of the invention to the host in an amount ofabout 10 to about 500 micrograms antigen per kilogram of body weight,preferably about 50 to about 100 micrograms antigen per kilogram of bodyweight. The proteins and vaccines of the invention can be administeredtogether with a physiologically acceptable carrier. For example, adiluent, such as water or a saline solution, can be employed.

Reference is made herein to proteins of the invention. The proteins ofthe invention include, for example, polypeptides that are notglycosylated. The proteins can be prepared using conventionaltechniques. For instance, glycosylation of the proteins in vivo can beblocked by tunicamycin, an antibiotic which inhibits N-linkedglycosylation of proteins (Schwartz et al., 1976; Kornfeld and Kornfeld,1985). Alternatively, glycoproteins of the invention can bedeglycosylated by β-N-acetylglucosaminase H (endo H), which cleaves highmannose-type oligosaccharide chains (Tarentino et al., J. Biol. Chem.,249: 818-824, 1974).

In summary, proteins and glycoproteins, which are precursors of HIV-2and SIV envelope glycoprotein and the dimeric form of the transmembraneglycoprotein, have now been identifed. In addition to providing usefultools for detection of antibodies to the retrovirus in humans and forraising neutralizing antibodies to HIV-2 in vitro and in vivo, thisinvention adds to the base of knowledge relating to immunodeficiencyactive proteins and glycoproteins of the AIDS viruses.

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What is claimed is:
 1. An isolated immune complex comprising a proteinand an antibody that binds with said protein, wherein the protein isselected from the group consisting of gp80 of HIV-2 and gp65 of SIV,wherein said gp80 is a glycoprotein having an apparent molecular weightof 80 kDa, as determined by SDS-PAGE, and further wherein said gp65 is aglycoprotein having an apparent molecular weight of 65 kDa, asdetermined by SDS-PAGE.
 2. The immune complex of claim 1, wherein theantibody, protein, or both the antibody and protein, are labeled with animmunoassay label selected from the group consisting of radioisotopes,enzymes, fluorescent labels, chemiluminescent labels, and chromophorelabels.
 3. An isolated immune complex comprising a peptide and anantibody that binds with said peptide, wherein the peptide has thefollowing sequence: VTAIEKYLQDQARLNSWGCAFRQVCH.
 4. The immune complex ofclaim 3, wherein the antibody, peptide, or both the antibody andpeptide, are labeled with an immunoassay label selected from the groupconsisting of radioisotopes, enzymes, fluorescent labels,chemiluminescent labels, and chromophore labels.
 5. An immunogeniccomposition comprising an amount of gp80 protein of humanimmunodeficiency virus type 2 (HIV-2) sufficient to induce an immuneresponse and a pharmaceutically acceptable carrier.
 6. The immunogeniccomposition of claim 5 further comprising one or more proteins of HIV-2selected from the group consisting of gp125, gp140, and gp300 of HIV-2.7. A composition comprising at least one antigen selected from the groupconsisting of gp80 protein of HIV-2 and gp65 of SIV.
 8. The compositionof claim 7, further comprising one or more proteins selected from thegroup consisting of gp125, gp140, and gp300 of HIV-2.